Nearly 90% of the protein in the retinal disc membrane is rhodopsin, a 38 kd integral membrane glycoprotein whose function is the transduction of electrical energy during visual excitation of photoreceptor cell outer segments. In this project, the membrane topology of bovine rhodopsin will be studied. Using a cDNA clone that contains the entire coding region for opsin, we will use an in vitro coupled transcription-translation system to synthesize radiolabeled gene product. This protein will then be assembled into microsomal membranes and analyzed to determine if the in vitro synthesis and assembly system faithfully mimics in vivo events. The intramembrane topology of opsin will be assessed by proteolytic digestion experiments as well as by a novel immunochemical approach that will use as probes antibodies produced to specific intramolecular segments in various amino- and carboxy-terminal regions of opsin. Our immediate goal is to insert opsin cDNA into a high copy vector (SP6), establish conditions for in vitro coupled transcription-translation and prepare topologically "correct" opsin-containing microsomal membranes. If successful, these pilot studies will form the basis for long-term studies that will use deletion mutants to assess the role of various regions of the opsin molecule during its insertion into membranes. For example, the intradiscal amino terminus may be deleted from opsin to assess the possible role of this region in mediating membrane translocation. If successful, these studies will permit a detailed analysis of the molecular topology of rhodopsin and the mechanisms involved in its membrane insertion. Such studies will also make it possible, for the first time, to study at the molecular level those fundamental properties of an integral membrane protein that are important for insertion into, and proper orientation within, the plasma membrane. These long-term goals will be accomplished by constructing plasmids containing opsin DNA with various segments of the molecule deleted and then assessing the effects of these deletions on opsin structure, topology and functions. In a more clinical context, these studies may provide a novel approach to understanding the retinal dystrophies (e.g. hereditary retinal degeneration). If there are defects in the synthesis or membrane organization of rhodopsin in such diseases, this experimental system may permit a more detailed analysis of possible mechanisms operating during retinal degeneration.